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Method for producing low-temperature hydrogen-amino oxidase from heterotrophic nitrification acinetobacter L7 of Harbin Institute of Technology, and separation and purification method thereof

A technology of hydroxylammonium oxidase and heterotrophic nitrification, which is applied in the field of enzyme production and can solve the problem that ammonia nitrogen cannot be effectively removed at low temperature

Active Publication Date: 2015-05-20
HARBIN INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem that the existing low-temperature ammonia nitrogen cannot be effectively removed, and provide a method for producing low-temperature hydroxylammonium oxidase by Heterotrophic Nitrifying Acinetobacter L7 of Harbin Institute of Technology

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  • Method for producing low-temperature hydrogen-amino oxidase from heterotrophic nitrification acinetobacter L7 of Harbin Institute of Technology, and separation and purification method thereof

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specific Embodiment approach 1

[0017] Specific embodiment one: the method for producing low-temperature hydroxylammonium oxidase by Heterotrophic Nitrifying Acinetobacter L7 of Harbin Institute of Technology in this embodiment is carried out according to the following steps:

[0018] Inoculate Harbin Institute of Technology Heterotrophic Nitrifying Acinetobacter L7 (Acinetobacter Heteronitrifihit L7) strain into the enzyme-producing medium with an inoculation amount of 2%, and culture it at 160rpm / min and 15°C for 14-24h, and the Harbin Institute of Technology Heterotrophic Nitrifying Acinetobacter is completed. L7 produces low-temperature hydroxylamine oxidase; the components of the enzyme-producing medium are as follows: NH 4 Cl0.1g / L, CH 3 CH 2 ONa1.0g / L, NH 2 OH0.7g / L, MgSO 4·7H 2 O0.05g / L, K 2 HPO 4 0.2g / L, NaCl0.12g / L, CuSO 4 ·5H 2 O0.01g / L, NaMoO 4 2H 2 O0.05g / L, BaCl 2 0.01g / L, MnSO 4 4H 2 O0.01g / L, FeSO 4 0.01g / L, K[Fe(CN) 6 ]0.01g / L, CtyC0.02g / L and the remainder of distilled water,...

specific Embodiment approach 2

[0022] Specific embodiment two: the difference between this embodiment and specific embodiment one is that CH in the components of the enzyme-producing medium is 3 CH 2 ONa can be replaced by propionate or butyrate. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0023] Specific embodiment three: In this embodiment, the method for further separation and purification of low-temperature hydroxylammonium oxidase produced by Heterotrophic Nitrifying Acinetobacter L7 of Harbin Institute of Technology is carried out according to the following steps:

[0024] 1. Inoculate the strain of Acinetobacter Heteronitrifihit L7 (Acinetobacter Heteronitrifihit L7) of Harbin Institute of Technology into the enzyme-producing medium with an inoculum size of 2%, culture at 160rpm / min and 15°C for 14-24h, and then centrifuge at 6000rpm for 5min to collect bacteria;

[0025] 2. Resuspend the bacteria with 10mmol / L, pH8.5 Tris-HCI buffer, place them in an ice-water bath for ultrasonic disruption for 15 minutes, then use lysozyme to disrupt the cells, and divide the collected bacteria into The cytoplasm, periplasm and cell membrane are three parts, and then through osmosis, the extracellular matrix without lysozyme is prepared;

[0026] 3. Use DEAE-CL6B ion e...

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Abstract

The invention discloses a method for producing low-temperature hydrogen-amino oxidase from heterotrophic nitrification acinetobacter L7 of Harbin Institute of Technology, and a separation and purification method of the low-temperature hydrogen-amino oxidase, and relates to a method for enzyme production and a separation and purification method of enzymes. The method comprises the following steps of: producing enzymes, namely, inoculating L7 strains into an enzyme production fluid nutrient medium to be cultured, separating and purifying, namely, firstly, inoculating the L7 strains into the enzyme production fluid nutrient medium to be cultured, and collecting thallus, secondly, crushing the thallus and preparing an extracellular matrix without lysozyme, and thirdly, separating the extracellular matrix, balancing, eluting, carrying out chromatography, carrying out electrophoresis, and collecting monomer proteins. The L7 strains inoculated into the enzyme production fluid nutrient medium are high in enzyme yield and stable in activity, and the enzyme activity is the highest and the enzyme yield is the highest at 15 DEG C. According to the separation and purification method disclosed by the invention, the produced low-temperature hydrogen-amino oxidase is a monomer protein with a molecular weight of 60.4KDa, the enzyme activity is stable at 2-25 DEG C when the pH value is 7.5 to 8.5, and ammonia nitrogen in low-temperature water is effectively eliminated.

Description

technical field [0001] The present invention relates to methods for producing enzymes. Background technique [0002] Biological denitrification is currently recognized as the most effective and promising technology for removing ammonia nitrogen pollution in water, and it is also a research focus and hotspot in the field of water pollution control. Whether it is autotrophic nitrifying bacteria or heterotrophic nitrifying bacteria, in the process of ammonia nitrogen degradation, it will be affected by temperature. A decrease in ambient temperature will result in poor ammonia nitrogen degradation, especially when the temperature is lower than 15°C, microorganisms stop growing and ammonia nitrogen cannot be degraded. The winter water temperature in northern my country is usually lower than 10°C. Taking Heilongjiang Province as an example, the lowest winter water temperature is below 2°C. The low temperature makes the removal of ammonia nitrogen a difficult problem. [0003] Th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/06C12R1/01
Inventor 李伟光张多英黄晓飞秦雯
Owner HARBIN INST OF TECH